11) Identification

• Accurate identification of microbes is essential for research and industry (e.g. clinical microbiology to food production)

• Identification is a part of taxonomy

Indirect methods (conventional methods) -> isolation and culture of microorganisms and the determination of their various phenotypic characteristics

Direct methods (culture-independent) -> used to identify specific microbes in a mixed population as well as identify non- culturable microbes

Phylogenetic classification

Microscopy methods

Biochemical techniques

Molecular Methods

Phenotypic:

• labor and time intensive

• often ambigous (esspecially on the strain level)

Genomic:

• Most medically important bacteria can be grown on agar plates

• Give rise to colonies

• Each colony is assumed to derive from a single bacterium

• Colony morphology gives information about the bacterium

• Media has to contain the right type of nutrients for the respective species

• correct (atmosphoric) conditions are needed

Selective medium

• allows selective organisms to grow and inhibits the growth of others

• CCEY, tellurite agar, MacConkey agar

Differential medium

• can differentiate closly related organisms

• uses special dyes or chmicals to charaterize organisms

• blood agar, chromogenic media

• Distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet dependent on the thickness of the peptidoglycanwall

• Can be used directly on some clinical material

• Good for early detection

Ablauf:

1. Make a smear of cells on a microscope slide

2. Allow the smear to dry, then heat fix it

3. Flood the smear with crystal violet

4. Add Gram’s iodine

5. Add decolorizer (alcohol or acetone)

6. Add counterstain (e.g. safranin)

7. Wash off reagents with water 8. Viewed smear under oil immersion lens of light microscope

• difficult if bacterium has a complicated morpholoy

• another nethod is always needed to comfim identity

• poor sensitivity

Used to identify acid-fast microbes

1. Make a smear of smear on a microscope slide

2. Allow the smear to dry, then heat fix it

3. Pour carbol fuschin over smear

4. Heat gently until fumes appear; do not overheat

5. Leave to stand for 5 minutes

6. Pour over 20 % sulfuric acid, wait for 1 minute and keep repeating step until the slide appears light pink in colour, then wash with water

7. Pour over methylene blue, wait for 2 minutes, wash with water

8. Air dry slide and examine under oil immersion lens of light microscope

Lactose +/-

Fast/Slow fermenter

Oxidase+/-

strict anaerob

Urease+/-

Catalase

• quick, cheap and simple

• tests presence of ctalase

• Present in aerobes and some facultative bacteria (Staphylococcus)

Coagulase

• after positive gram ans catalase test -> pathongenic Staphylococcus

• acts by a thrombinase-like action

• A coagulase-positive strain of Staphylococcus will clot plasma

Oxidase

• only for gram-

• detects presence of cytochrome oxidase

• oxidase + Neisseria, Pseudomonas, Vibrio, Helicobacter, Campylobacter

• Allows fast identification

• Standardised, miniaturized phenotypic tests for a range of micro-organisms

• Range of kits for different organisms

• Has been the “gold standard” especially in clinical microbiology

• A variety of tests can be used including restricted-based digestion methods

• Simultaneous detection and identification of pathogen

• Molecular tests can be done from viable and non-viable cells

• Becoming more widely used to detect/identify bacteria or to type them

• Some knowledge of the target genome is required

• 1500 nt long, long enough for informative comparative and phylogenetic analyses

• Has conserved and variable regions

• Comparative analyses (BLAST, EzTaxon)

• higher sensitivity and accuracy copared to normal PCR

• ability to monitor DNA amplification in real-time through fluorescence intensity → negating need for any post-PCR detection techniques

• can be quantitative or semi - quantitative

RAPD-PCR:

• Uses universal random primers as alternatives to gene specific molecular marker identification

• Useful when analysing a large number of samples of diverse species and without any prior genetic information

• suffers from poor reproducibility between laboratories because of inconsistent PCR amplification conditions

RFLP

• PCR-based method employs restriction enzymes, which can recognise and cut amplified DNA (PCR product) into DNA fragments of different lengths

• different fragments are separated to generate a unique pattern of bands for each bacterial strain

• slow with poor reproducibility

PFGE

• Method of separating large fragments of DNA

• Useful for characterising and typing bacteria for epidemiological studies

• used to be the current “gold standard” fingerprinting method

• Analysis of the entire bacterial genome

• Provides insights into bacterial typing and evolutionary lineages

• Sange sequnecing, illumina, PacBio, NanoPore

• Process:

  • Extraction

  • Shearing

  • Library Preparation

  • Library Sequencing

  • Sequencing Analysis

• multiple immunological tests to identify different types of bacteria

• rely on antigen binding to antibody

• Antigen (Ag)

• Antivody (Ab)

  • classes IgG, IgM, IgA, IgD, IgE

  • combine with specific antigen

• Ag-Ab reactions can be used for bacterial identification

• Agglunation

  • Antigen reacts with its corresponding antibody → visible clumping of bacterial cells

  • easily visible precipitate

  • used for initial confirmation (salmonella, vibrio cholerae)

Enzyme-linked immunosorbent assay

• technique to detect an antigen or antibody

• Types: Direct, Indirect, Sandwich, Competitive, Multiplex

• Sandwich:

  • Antibody -> Antigen -> Antibody -> Substrate

  • Absorbance relates to concentration

• Competitive:

  • Antigen -> specific Ab + sample + enzyme-conjungated secondary Ab + substrate

  • used for quantification

Lateral flow immunoassays

• rapid, easy, durable stability, low cost, no additional equipment

• aka immunochromatographic assay

• two types

  • double antibody sandwich assays

  • competitive assays

Mass Spectrometry

• Advantages: speed, reduced costs, simplicity, applicability for a wide range of bacteria

• Several ionization and separation techniques can be coupled with MS

FTIR

• Chemical and label-free procedure

• fast, versatile, non-invasive, easy to perform

• only small amount of sample required

• Possible to obtain, in a single measurement lipids, proteins, carbohydrates, and nucleic acids

MALDI-TOF-MS

• ionization of microbial cells with short laser pulses and then accelerating the particles in a vacuum system using an electric field

• a specific molecular fingerprint in the form of a spectra profile is obtained

• fingerprint can be compared to a database